Measurement of oxidative stress and antioxidant status in acute lymphoblastic leukemia patients

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Clinical Biochemistry 41 (2008) 511 – 518

Measurement of oxidative stress and antioxidant status in acute lymphoblastic leukemia patients Vanessa Battisti a , Liési D.K. Maders a , Margarete D. Bagatini a , Karen F. Santos a , Rosélia M. Spanevello b , Paula A. Maldonado a , Alice O. Brulé c , Maria do Carmo Araújo c , Maria R.C. Schetinger a , Vera M. Morsch a,⁎ a

b

Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, Campus Universitário, 97105-900 Santa Maria, RS, Brazil Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcellos, 2600-Anexo, 90035-003, Porto Alegre, RS, Brazil c Hospital Universitário de Santa Maria-HUSM, Centro de Ciências da Saúde, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil Received 1 October 2007; received in revised form 27 December 2007; accepted 28 January 2008 Available online 15 February 2008

Abstract Objectives: To evaluate the oxidative status and antioxidant defense in patients with acute lymphoblastic leukemia (ALL). Design and methods: We measured concentrations of plasmatic thiobarbituric acid reactive substances (TBARS), serum protein carbonylation, whole blood catalase (CAT) and superoxide dismutase (SOD) activities, as well as the plasmatic and erythrocyte thiol levels and serum vitamin E concentration. This study was performed on 80 children with ALL divided into 4 groups: just diagnosed, remission induction, remission maintenance and out-of-treatment. Results: TBARS levels and serum protein carbonylation were higher in ALL patients than in controls and reduced levels of antioxidants were found in these patients. Conclusion: These findings may indicate a possible link between decreased antioxidants and increased levels of cells alterations due to oxidative damage, supporting the idea that there is a persistence of oxidative stress in acute lymphoblastic leukemia. © 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Acute lymphoblastic leukemia (ALL); Oxidative stress; Antioxidant system; TBARS; Protein carbonylation; SOD; CAT; Thiols; Vitamin E

Introduction Leukemias originate from hematopoietic stem cells that lose the capacity to differentiate normally in mature blood cells at different stages of their maturation and differentiation [1,2]. Acute lymphoblastic leukemia (ALL) is the most common cancer found in the pediatric population and it accounts for more than 50% of the hematopoietic malignancies in this age group [3,4]. In contrast, ALL is a relatively rare leukemia subtype in adults, accounting for only 2–3% of hematopoietic malignancies [4]. ALL is a disease characterized by uncontrolled proliferation and maturation arrest of lymphoid progenitor cells in bone ⁎ Corresponding author. Fax: +55 55 3220 8978. E-mail address: [email protected] (V.M. Morsch).

marrow resulting in an excess of malignant cells [5]. The lymphoblasts replace the normal marrow elements, resulting in a marked decrease in the production of normal blood cells. ALL is a disorder caused by an abnormal expression of genes, which is usually a result of chromosomal translocations [6]. The disease can be originated from lymphoid cells of different lineages, giving rise to B- or T-cell leukemias or sometimes mixed-lineage leukemia [7]. It is a curable disease with an expected long term survival rate of at least 70%, when treated with modern therapeutic regimens. In general, ALL standard treatment protocols consist of induction and maintenance remission with chemotherapeutic drugs [8]. It is well recognized that oxidants play a role in several stages of carcinogenesis [9]. Production of reactive oxygen species (ROS) is an inevitable result in cells that use aerobic

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V. Battisti et al. / Clinical Biochemistry 41 (2008) 511–518

metabolism for energy production [10]. ROS are known to play a dual role in biological systems, since they may be either harmful or beneficial to living systems. Furthermore, oxidative stress may inhibit or promote apoptosis and until necrosis, depending on the intensity of the stimulation. Beneficial effects of ROS involve physiological roles in cellular responses to noxia, as for example in the defense against infectious agents and in the function of a number of cellular signaling systems [11]. In contrast, at high concentrations, ROS can be important mediators of damage to biomolecules such as DNA, proteins, and lipids, leading to cellular dysfunction and cell death. Accumulation of such molecules causes noxious effects on individuals, resulting in diseases such as hematopoietic malignancies [12]. The first biological molecules for oxidative damage in cells are proteins and their side chains can be carbonylated by reactive carbonyl compounds [13]. In addition, oxidative damage in lipids leads to the formation of products such as malondialdehyde (MDA) [14]. The effect of reactive species is balanced by the antioxidant action of non-enzymatic antioxidants, as well as by antioxidant enzymes. Antioxidant defenses are extremely important as they represent the direct removal of free radicals (pro-oxidants), providing maximal protection for biological sites [11]. The most efficient enzymatic antioxidants involve superoxide dismutase (SOD) and catalase (CAT). SOD is an antiU oxidant enzyme that catalyzes the dismutation of O2− to O2 and to the less-reactive species hydrogen peroxide (H2O2) protecting cells from injury induced by free radicals [15]. Additionally, the enzyme CAT very efficiently promotes the conversion of H2O2 to water and molecular oxygen [14]. Non-enzymatic antioxidants include thiol antioxidants and vitamin E. Nonprotein thiols have a variety of functions in bioreduction and detoxification processes [16]. The main antioxidant function of vitamin E is the protection against lipid peroxidation [17]. Information about the activities of antioxidant enzymes is conflicting in patients with cancer and studies on leukemia patients are rare [18]. Moreover, the levels of oxidative damage and antioxidant defenses have not been investigated in children just diagnosed with ALL as compared to those in the different stages of treatment and after therapy. In this work, we studied the oxidative profile in ALL patients in these groups, through the verification of main enzymatic antioxidant defenses (CAT and SOD) and non-enzymatic antioxidants (thiols and vitamin E). Moreover, we determined the intensity of biological damage caused by free radicals in lipid and protein through the measurement of lipid peroxidation and the levels of protein carbonylation.

diabetes, parasitosis or any immune dysfunction. Also, the controls used in this study had normal leukocytes and other blood cell counts and made no use of pharmacological therapy. Patients included in this study received the diagnosis for ALL, which is based on the following findings: leukocyte count, age, involvement of tissues other than bone marrow, immunophenotyping and responsiveness to the treatment. Patients were diagnosed and treated according to the GBTLI LLA-99 protocol [19] defined by the Brazilian Group of Childhood Leukemia Treatment. The treatment with chemotherapeutic agents consisted of two phases: remission induction and remission maintenance. The first phase starts with induction therapy, using multiple chemotherapeutic drugs, followed by consolidation. Remission maintenance is a long phase, in which patients undergo up to 84 weeks of chemotherapeutic agents. After these phases, patients have follow-ups with periodic laboratorial exams to eliminate the possibility of recurrence. Taking this into account, we selected four patient groups: The first group was composed of 10 children (ages ranged from 3–17 years) just diagnosed with ALL who had not received any therapy preceding the blood sampling. The second group was composed of 20 patients (ages ranged from 3–19 years) undergoing treatment in the first phase, remission induction. The third group was composed of 25 patients (ages ranged from 3–23 years) in the second phase of treatment, called remission maintenance. 25 patients out-of-treatment (ages ranged from 4–20 years) constituted the fourth group. Consent was given by family members of all the patients included in this work. The Human Ethics Committee from the Federal University of Santa Maria approved the protocol under number 51/06. The patients' general characteristics are shown on Table 1. Sample collection The blood was collected in vaccutainer tubes without an anticoagulant system, centrifuged at 5000 rpm for 10 min, the precipitate was discarded and the serum was used to determine the protein carbonyl and the vitamin E contents. CAT and SOD activities were determined using whole blood collected in citrated vaccutainer tubes and diluted in a 1:10 in saline solution. For plasmatic thiobarbituric acid reactive substances (TBARS) and non-protein thiols, the blood was collected using EDTA as anticoagulant. The sample was then centrifuged (5000 rpm for 10 min) and the plasma was used to determine MDA and thiols and the erythrocytes were used only to verify thiol levels. Samples were obtained from November 2006 to May 2007.

Methods

Hematological determinations

Patients

Quantitative determinations of blood cells obtained by venipuncture were performed using a Coulter-STKS analyzer (Miami, USA).

The sample consisted of 80 acute lymphoblastic leukemia (ALL) patients recently diagnosed and under treatment at the Oncology-Hematology Laboratory—Hospital of the Federal University of Santa Maria. The controls consisted of 50 healthy volunteers with ages and social conditions similar to those of the patients. They presented no acute or chronic diseases such as

Carbonylation of serum protein The carbonylation of serum proteins was determined by a modified Levine's method [20]. Firstly, from 1 mL of serum, the

V. Battisti et al. / Clinical Biochemistry 41 (2008) 511–518 Table 1 General characteristics of patients

Catalase (CAT) and superoxide dismutase (SOD) activities

General characteristics

Just diagnosed

Remission induction

Remission maintenance

Out-oftreatment

Age range (years)

3–17

3–19

3–23

4–20

Sex

5 5 5 5 5

12 8 8 12 13 7

15 10 13 12 21 4

15 10 12 13 19 6

Risk Type

Male Female LR HR B T5

Hematological features in blood WBC (103 mm3) Lymphocytes (103 mm3) Hb (g/dL) Platelets (103 mm3) Erythrocytes (millions/mm3) Blasts (103 mm3)

66.0 ± 38.1a 9.7 ± 4.8a

4.9 ± 1.2b 1.3 ± 0.2b

3.9 ± 0.3b 1.1 ± 0.1b

7.0 ± 0.4b 2.4 ± 0.1b

7.2 ± 0.8a 56.6 ± 15.5a

8.0 ± 0.6a 191.4 ± 32.9b 2.6 ± 0.2a

11.5 ± 0.2b 230.6 ± 19.5b 3.67 ± 0.1b

13.2 ± 0.2c 250.0 ± 16.9b 4.67 ± 0.3c

–

–

–

2.5 ± 0.3a 27.7 ± 13.1

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HR: high recurrence risk, LR: low recurrence risk, WBC: white blood cell count, Hb: hemoglobin. Data are presented as mean ± SEM. Duncan's multiple range test: groups that show different letters are statistically different (p b 0.05).

The determination of CAT activity was carried out in accordance with a modified method of Nelson and Kiesow [22]. This assay involves the change in absorbance at 240 nm due to CAT dependent decomposition of hydrogen peroxide. An aliquot (0.02 mL) of blood was homogenized in potassium phosphate buffer, pH 7.0. The spectrophotometric determination was initiated by the addition of 0.07 mL in an aqueous solution of hydrogen peroxide 0.3 mol/L. The change in absorbance at 240 nm was measured for 2 min. CAT activity was calculated using the molar extinction coefficient (0.0436 cm2/μmol) and the results were expressed as picomoles per milligram protein. SOD activity measurement is based on the inhibition of the radical superoxide reaction with adrenalin as described by Mc Cord and Fridovich [23]. In this method, SOD present in the sample competes with the detection system for radical superoxide. A unit of SOD is defined as the amount of enzyme that inhibits by 50% the speed of oxidation of adrenalin. The oxidation of adrenalin leads to the formation of the colored product, adrenochrome, which is detected by spectrophotometer. SOD activity is determined by measuring the speed of adrenochrome formation, observed at 480 nm, in a reaction medium containing glicine-NaOH (50 mM, pH 10) and adrenalin (1 mM). Determination of non-protein thiols

proteins were precipitated using 0.5 mL of 10% trichloracetic acid (TCA) and centrifuged at 5000 rpm for 5 min discarding the supernatant. One half milliliter of 10 mmol/L 2.4-dinitrophenylhydrazine (DNPH) in 2 mol/L HCl was added to this protein precipitate and incubated at room temperature for 30 min. During incubation, the samples were mixed vigorously every 15 min. After incubation, 0.5 mL of 10% TCA was added to the protein precipitate and centrifuged at 5000 rpm for 5 min. After discarding the supernatant, the precipitate was washed twice with 1 mL of ethanol/ethylacetate (1:1), centrifuging out the supernatant in order to remove the free DNPH. The precipitate was dissolved in 1.5 mL of protein dissolving solution (2 g SDS and 50 mg EDTA in 100 mL 80 mmol/L phosphate buffer, pH 8.0) and incubated at 37 °C water bath for 10 min. The color intensity of the supernatant was measured using a spectrophotometer at 370 nm against 2 mol/L HCl. Carbonyl content was calculated by using the molar extinction coefficient (21 × 103 1/mol cm) and the results were expressed as nanomoles per milligram protein. Determination of lipid peroxidation Lipid peroxidation was estimated by measuring TBARS in plasma samples according to a modified method of Jentzsch et al. [21]. Briefly, 0.2 mL of serum was added to the reaction mixture containing 1 mL of 1% ortho-phosphoric acid, 0.25 mL alkaline solution of thiobarbituric acid-TBA (final volume 2.0 mL) followed by 45 min heating at 95 °C. The results were expressed as nanomole MDA per milliliter of plasma.

Non-protein thiols were assayed in plasma and erythrocytes by the method of Ellman [24]. Aliquots (0.1 mL) of plasma were added to a phosphate buffer 0.3 mol/L (0.85 mL), pH 7.4 and the reaction was read at 412 nm after the addition of 10 mM 5-5V-dithio-bis(2-nitrobenzoic acid) (DTNB) (0.05 mL). Results were expressed as μmol/mL of plasma. Aliquots of erythrocytes (0.3 mL) were hemolyzed with 10% Triton X-100 (0.1 mL) and, after 10 min, precipitated with 0.2 mL of 20% TCA. After centrifugation at 5000 rpm for 10 min, the supernatant aliquots reacted with 50 μL of DTNB (10 mM) and the reaction product was read at 412 nm. Results were expressed as μmol/mL of erythrocyte. Serum vitamin E quantification Serum vitamin E was estimated by a modified method of Hansen and Warwick, [25]. In a cover tube, 140 μL of Milli-Q water (Millipore, Bedford, MA, USA) was added to 20 μL of butylated hydroxytoluene 10 mM (BHT), 140 μL of sample and 2.1 mL of ethanol solution (66%). After this, it was vortexmixed for 10 s and 3.5 mL of n-hexane was added and mixed for 1 min. It was then centrifuged at 1800 g for 10 min and 3 mL of superior phase transferred to fluorimeter cuvettes and the vitamin E was measured in the fluorimeter: excitement: 295 nm; emission: 340 nm. All samples were analyzed in duplicate. Calibration curves with α-tocoferol (Sigma-Aldrich Inc, USA) were used to determine the concentration, following the same procedure for the samples.

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Fig. 1. Protein carbonylation content in serum of ALL patients. Patients were divided into: just diagnosed (JD, n = 10), remission induction (RI, n = 18), remission maintenance (RM, n = 25) and out-of-treatment (OT, n = 20) groups. Controls consisted of 40 healthy subjects. Each column represents mean ± S.D. Duncan's multiple range test: groups that show different letters are statistically different (p b 0.05).

Protein determination Protein was measured by the method of Bradford [26] using bovine serum albumin as standard. Statistical analysis Statistical analysis was done by the commercial SPSS package for Windows©. All the data are expressed as the mean ± standard error. Data were analyzed statistically by one-way ANOVA followed by the Duncan's multiple test. Differences were considered significant when the probability was p b 0.05.

Fig. 3. CAT activity in total blood of ALL patients. Patients were divided into: just diagnosed (JD, n = 10), remission induction (RI, n = 20), remission maintenance (RM, n = 22) and out-of-treatment (OT, n = 17) groups. Controls consisted of 40 healthy subjects. Each column represents mean ± S.D. Duncan's multiple range test: groups that show different letters are statistically different (p b 0.05).

the patients just diagnosed. White blood cell count, including lymphocytes was elevated in the just diagnosed patients and platelets count was diminished in these patients. Hemoglobin and erythrocytes were decreased in the just diagnosed and remission induction patients. Protein carbonylation Protein oxidation, determined by protein carbonyl content in serum samples from the patients, is shown in Fig. 1. It can be observed that there was a significant difference between the patients and the controls. The protein carbonyl content was increased in the just diagnosed patients, in patients of both treatment phases and also in the out-of-treatment patients when compared to the controls (F(4,108) = 7.62; p b 0.001). Lipid peroxidation

Results Characteristics and hematological features in blood Table 1 presents the patient's general characteristics and blood cell count. We observe the presence of blast cells in

Fig. 2. TBARS levels in plasma of ALL patients. Patients were divided into: just diagnosed (JD, n = 10), remission induction (RI, n = 20), remission maintenance (RM, n = 25) and out-of-treatment (OT, n = 22) groups. Controls consisted of 44 healthy subjects. Each column represents mean ± S.D. Duncan's multiple range test: groups that show different letters are statistically different (p b 0.05).

The lipid peroxidation results are shown in Fig. 2. Post-hoc comparisons by Duncan's test revealed that TBARS content was increased in the just diagnosed patients and in the remission induction patients in relation to controls and to the remission

Fig. 4. SOD activity in total blood of ALL patients. Patients were divided into: just diagnosed (JD, n = 10), remission induction (RI, n = 18), remission maintenance (RM, n = 25) and out-of-treatment (OT, n = 20) groups. Controls consisted of 40 healthy subjects. Each column represents mean ± S.D. Duncan's multiple range test: groups that show different letters are statistically different (p b 0.05).

V. Battisti et al. / Clinical Biochemistry 41 (2008) 511–518

Fig. 5. Non-protein thiol levels in plasma of ALL patients. Patients were divided into: just diagnosed (JD, n = 10), remission induction (RI, n = 19), remission maintenance (RM, n = 24) and out-of-treatment (OT, n = 21) groups. Controls consisted of 40 healthy subjects. Each column represents mean ± S.D. Duncan's multiple range test: groups that show different letters are statistically different (p b 0.05).

maintenance and out-of-treatment patients (F(4,116) = 8.94; p b 0.001), but the remission maintenance and out-of-treatment patients was also increased in relation to the controls. Antioxidant catalase (CAT) and superoxide dismutase (SOD) activities CAT activity in just diagnosed, remission induction and remission maintenance patients was reduced when compared to controls (F(4,104) = 16.09; p b 0.001). No significant difference was observed between the out-of-treatment patients and the controls (Fig. 3). Fig. 4 shows the SOD activity in ALL patients and controls. No statistically significant relationship was found between the remission maintenance and out-of-treatment patients and the controls, or between the remission induction and out-oftreatment patients. Nevertheless, the SOD activity was shown to be decreased in the just diagnosed and the remission induction patients in relation to the controls (F(4,103) = 4.87; p b 0.01).

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Fig. 7. Vitamin E content in serum of ALL patients. Patients were divided into: just diagnosed (JD, n = 10), remission induction (RI, n = 17), remission maintenance (RM, n = 20) and out-of-treatment (OT, n = 20) groups. Controls consisted of 40 healthy subjects. Each column represents mean ± S.D. Duncan's multiple range test: groups that show different letters are statistically different (p b 0.05).

Levels of non-protein thiols in plasma and erythrocytes Post-hoc comparisons made by Duncan's test revealed that the levels of non-protein thiols in plasma were reduced in the remission induction, remission maintenance and just diagnosed patients, (F(4,119) = 6.43; p b 0.001), while it was not significantly different for the out-of-treatment patients in relation to the controls (Fig. 5). Thiol levels in erythrocytes were also decreased in the remission maintenance patients when compared to the controls (F(4,108) = 7.95; p b 0.001). However, there was no significant difference between the just diagnosed, remission induction and out-of-treatment patients in relation to the controls (Fig. 6). Serum vitamin E content Serum vitamin E content was reduced both in the just diagnosed and remission induction patients, while it was not significantly different for the remission maintenance and out-oftreatment patients in relation to controls (F(4,102) = 5.60; p b 0.001). No statistically significant relationship was found between the remission maintenance patients and the other groups (Fig. 7). Discussion

Fig. 6. Non-protein thiol levels in erythrocytes of ALL patients. Patients were divided into: just diagnosed (JD, n = 8), remission induction (RI, n = 17), remission maintenance (RM, n = 25) and out-of-treatment (OT, n = 23) groups. Controls consisted of 50 healthy subjects. Each column represents mean ± S.D. Duncan's multiple range test: groups that show different letters are statistically different (p b 0.05).

ALL is more prevalent, and has a better prognosis, in children. This disease was diagnosed on the basis of clinical history, physical examination and complete hemogram. Patients with ALL can have a high, normal, or low white blood cell count. The lymphoblasts replace the normal marrow elements, resulting in a marked decrease in the production of normal blood cells. Consequently, anemia, thrombocytopenia, and neutropenia occur to varying degrees. Extensive evidence has shown that disturbances of oxidative stress metabolism are a common feature of transformed tumor cells [27]. Both alterations of antioxidants and increases in the production of oxygen reactive species have been reported [28]. As a result, higher rates of lipid peroxidation and protein lesions have been found in the majority of neoplastic tissues. Furthermore, altered levels of antioxidant enzymes (SOD, CAT) and non-

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enzymatic antioxidants (thiols, vitamin E) are evident in many human cancers [29]. There is a relationship between leukemia and oxidative stress. Leukemic cells produce higher amounts of reactive oxygen species (ROS) than non-leukemic cells as they are under a repeated state of oxidative blockade [30]. However, discrepancies as to the reported changes of enzymatic activities in these patients are reflected in the scientific literature. Moreover, a study that analyzes the oxidative profile in children just diagnosed with ALL in comparison to that of the different stages of treatment has not been found in the literature. Other studies evaluated antioxidant status in children with newly diagnosed ALL and during the first months of chemotherapy [31,32], but not with patients out-of-treatment. In this context, we have examined the activities of antioxidant enzymes (SOD, CAT) as well as thiol, vitamin E and MDA levels and protein carbonylation in the blood of just diagnosed ALL patients as compared to those in the different stages of treatment and after therapy. In the present study, the levels of protein carbonylation and TBARS contents were shown to be increased in the just diagnosed patients, in the two treatment phases and in the outof-treatment patients when compared with the control group (Figs. 1 and 2). These results are in accordance with the increase of TBARS levels in the serum of patients with chronic leukemia [30] and acute lymphoblastic leukemia [33] and of bone marrow transplant recipients [34]. On the other hand, Devi el al. [18] and Er et al. [35] showed that plasma lipid peroxidation products in untreated leukemia patients and in patients with acute myeloid leukemia were in the normal range and Popadiuk et al. [36] demonstrated that in children with malignant bone tumors no alterations in the level of protein carbonylation were found. We can suggest that the increase of oxidative lesions seems not to be a result of the treatment with chemotherapeutic agents but may be involved with the pathogenesis of leukemia, since in the non treated patients, the levels were more increased than in those in treatment. Moreover, protein carbonylation may be an irreversible lesion as this parameter was high in the out-oftreatment group. It has been suggested that oxidative damage accumulates in biological molecules during aging and that oxidative stress is relevant to the aging process. However, the antioxidant capacity of tissues decreases during aging. Thus, the oxidative profile changes with age. In this study, although all the groups have an age range of 3–23 years, this difference in age does not affect the results, since the values found for markers of oxidative stress were adequately homogeneous in all groups. The data reported in the literature concerning antioxidant enzymes in different human cancer types are controversial. In this study, it was demonstrated that SOD and CAT activities were decreased in ALL patients. CAT activity was reduced in just diagnosed patients and patients in both treatment groups. SOD activity was decreased in the just diagnosed and remission induction patients (Figs. 3 and 4). This phenomenon indicates a disturbance of the protective role of these enzymes against free radicals in ALL. These findings are in accordance with earlier studies of Oltra et al. [37] who confirmed decreased SOD and

CAT activities in the lymphocytes of chronic lymphocytic leukemia (CLL) patients. The results are also in agreement with the reports of Sentuërker et al. [38], who demonstrated reduced CAT and SOD activities in the lymphocytes of ALL patients, and Madej et al. [39] who found a decreased activity of these enzymes during the development of the leukemic process in mice. However, Nishiura et al. [40] reported elevated serum SOD activity in acute leukemia and indicated that regression of the leukemia was accompanied by a decrease in the serum level of SOD. However, taken together, these findings suggest that there are alterations in the enzymatic antioxidant defenses, which can interfere in the direct removal of free radicals (prooxidants) and in the protection for biological sites. The cumulative production of free radicals during either endogenous or exogenous insults is common for many types of cancer cells and is linked with the altered redox regulation of cellular signaling pathways. Oxidative stress induces a cellular redox imbalance which has been found to be present in various cancer cells. The effect of reactive oxygen and nitrogen species is balanced by antioxidant enzymes such as CAT and SOD. In this context the impaired antioxidant role of CAT and SOD may support the accumulation of free radicals. Alternatively, it is possible that the antioxidant system is impaired as a consequence of an abnormality in the antioxidative metabolism due to the cancer process. This effect could be enhanced by the characteristic increase in the production of H2O2 by the cancer cells [41]. On the other hand, the SOD inhibition by novel anticancer agents may be a beneficial effect, resulting from induced apoptosis of the leukemia cells through a free radicalmediated mechanism [42]. Another important aspect to be discussed is that, no significant difference was observed between the out-of-treatment and the control groups for CAT and SOD activity. We suggest that these findings may be a consequence of the amount of time passed after the treatment, demonstrating that the treatment was in fact efficient. With respect to the thiol levels in plasma and erythrocytes, our findings revealed that the levels in plasma were reduced in remission induction, remission maintenance groups and just diagnosed patients. The thiol content in erythrocytes was also decreased in the remission maintenance patients when compared to the controls (Figs. 5 and 6). Glutathione (GSH), the main cellular thiolic compound, has a variety of functions in bioreduction and detoxification processes. Silber et al. [43] reported GSH depletion in lymphocytes isolated from the blood of patients with CLL. On the other hand, Oltra et al. [37] observed higher GSH concentration in lymphocytes of CLL subjects. There are many publications that reveal the effect of cancer on the antioxidant system [44]. However, previous evidence for the role played by thiols, mainly GSH, in determining a prognosis in leukemia has been conflicting. The decrease in thiol levels may represent a depletion of this antioxidant due to high concentrations of H2O2 and other peroxides formed in tumor cells. Therefore, thiols levels are not sufficient to prevent oxidative stress in the affected cells. Serum vitamin E content was reduced in both just diagnosed and remission induction patients; however these levels returned

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to the normal values for the remission maintenance and out-oftreatment patients (Fig. 7). A similar result was shown by Singh et al. [45] who found significantly decreased serum vitamin E levels in chronic myeloid leukemia patients before starting treatment and increased vitamin E levels after treatment. These findings confirm that vitamin E is an important antioxidant that is altered in leukemia. As an antioxidant, vitamin E may inhibit cancer formation by scavenging reactive oxygen or nitrogen species and could be considered the major membrane-bound antioxidant employed by the cell [46]. However, the epidemiologic evidence supporting a link between vitamin E and cancer is limited, and intervention studies are scarce [47]. The results reported in this paper characterize the persistence of oxidative stress in ALL. Although reactive species are well recognized for playing a dual role as both deleterious and beneficial species, excessive accumulation of reactive species contributes to antioxidant depletion and dysfunction. In addition, oxidative stress induces lipid peroxidation and protein carbonylation by inactivating antioxidant enzymes. One important aspect to be discussed is that the biggest differences in the parameters analyzed were observed in just diagnosed patients in compared to controls subjects, demonstrating the relationship between these changes and the development of ALL. In the outof-treatment patients, levels of antioxidants returned to normal values, indicating the regression of disease as a result of the treatment. In conclusion, the present work provides evidence for the increased levels of oxidative damage and decreased levels of the antioxidant system in ALL patients, suggesting a possible link between these two important parameters in this type of cancer. Furthermore, we hope that our results represent an important contribution in the study of the oxidative profile in children just diagnosed with ALL as compared to those in the different stages of treatment and after therapy. More studies are necessary to confirm whether these alterations are the cause or the consequence of carcinogenesis. Acknowledgments The authors wish to thank all the ALL patients and the professionals at the Hematology-Oncology Laboratory (HUSM) for their support. This study was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Rio Grande do Sul (FAPERGS), Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and the Federal University of Santa Maria, RS, Brazil. References [1] Sinnett D, Labuda D, Krajinovic M. Challenges identifying genetic determinants of pediatric cancers — the childhood leukemia experience. Fam Cancer 2006;5:35–47. [2] Molica S, Vacca A, Levato D, Merchionne F, Ribatti D. Angiogenesis in acute and chronic lymphocytic leukemia. Leuk Res 2004;28:321–4. [3] Gaynon PS. Childhood acute lymphoblastic leukaemia and relapse. Br J Haematol 2005;131:579–87.

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